induction and overexpression of a 7 kDa proteins in E coli
(by Zhonglin.Chai from bakeridi.edu.au)
Thu Aug 19 17:34:00 EST 2010
Possibility for this small protein to run out of your gel? PAGE
condition? Do you have other method to detect the presence of your
Sent from my iPhone
On 20/08/2010, at 5:11 AM, "venkatramanan rao" <ventigers from gmail.com>
> Hello all,
> I want to do domain mapping of my protein of intrest for testing its
> interaction with a few predicted interactors. I have generated
> N-termius end and C-terminus end of my protein. I have cloned
> approx150 bp both the terminii in pET28a vector between Nde1 and EcoRI
> sites(both the constructs). I tried inducing the protein in BL-21DE3
> cells using but no success. The full length protein expresses normally
> without any problem in a different vector backbone(infact gives a big
> blob of pure protein in induced cells) . I am wondering what went
> wrong. I have sent my plasmid samples for sequencing and awaiting
> for results. The constructs were PCR amplified using Pfu
> polymerase(very less chances of errors in PCR product). Since I have
> done directional cloning chances of frameshift is also less. I tried
> for presence of inserts using both colony PCR and plasmid as template
> both methods show presence of template of correct size. I was
> wondering if anybody has expressed protein of similar size. Is it
> getting degraded. Please help me in this regard.
> Rao Venkatramanan G.
> Research Scholar
> Mumbai University campus,
> Kalina, Santacruz
> Methods mailing list
> Methods from net.bio.net
> This email has been scanned by the MessageLabs Email Security System.
This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed.
If you have received this email in error please promptly delete this email and notify the system manager.
This email has been scanned by the MessageLabs Email Security System.
For more information please visit http://www.messagelabs.com/email
More information about the Methods